Techniques in molecular biology and molecular medicine often rely on analysis of single biological molecules. Such techniques include DNA and RNA sequencing, polymorphism detection, detection of proteins of interest, detection of protein-nucleic acid complexes, and many others. The high sensitivity, high throughput and low reagent costs involved in single molecule analysis make this type of analysis an increasingly attractive approach for a variety of detection and analysis problems in molecular medicine, from low cost genomics to high sensitivity marker analysis.
Many techniques for single molecule analysis rely on immobilization of the molecule or complex of interest on a solid support, typically within an optical confinement reaction/observation region such as a zero mode waveguide Immobilization of a given molecule must be robust, since dissociation means that molecule is lost to further analysis.
Immobilization of biological molecules is frequently accomplished by capture through moieties such as biotin. Biotin is a cofactor that is covalently attached to several enzymes involved in the transfer of activated carboxyl groups. Biotin labeling of molecules not normally biotinylated can be used to label, detect, purify, and/or immobilize such molecules. These methods also rely upon proteins such as avidin or streptavidin, which bind very tightly and specifically to biotin. However, single molecule analysis imposes challenges not seen in analysis of bulk samples, since it relies on robust immobilization of individual molecules rather than of a population of such molecules.
Improved methods for immobilizing single molecules and complexes are therefore desirable. The invention described herein fulfills these and other needs, as will be apparent upon review of the following.